Part:BBa_K2271024:Experience

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After expression and correct localization to the peroxisome was validated we examined the function of roGFP2. We conducted an in vitro assay on fully oxidized and fully reduced roGFP2 and performed time measurements by subsequently adding  H₂O₂  and DTT to the protein extract.

      

In the beginning roGFP2 was either treated with 1 mM DTT reducing the sensor or with 1 mM  H₂O₂ to oxidize the protein supernatant. Later the complete oxidation/reduction was achieved through adding additional and DTT.

We could observe a functional sensor with a high dynamic range in the cytosolic and the PTS1 fused construct, which indicates high sensitivity. Further the  PTS1 Tag  does not seem to disturb the function of roGFP2(data not shown). The calibration was performed using the mid point calibration method, which was previously performed by assuming the midpoint potential to be at -280mV Schwarzländer  et al.(2008) .

      

405/485nm excitation ratio plotted against the oxidized proportion of roGFP2

Based on the nernst equation we were now able to calculate the redox potential of roGFP2 regarding the oxidation of roGFP2 Schwarzländer,  et al.(2008).

      

Oxidized roGFP2 proportion plotted against the redox potential of glutathione (mV)

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Using our calibrated sensor we could compare the redox states within strains which differ in metabolic physiology.

      

Cytosolic and peroxisomal 405/485 nm ratios of roGFP2 were obtained. We used a strong(+) and weaker(-) Promoter. Yeast were grown on yeast nitrogen dropout medium at pH 6.0.Cultures showed an OD₆₀₀ between 0.9 and 1.1.

Comparisons of the cytosolic and peroxisomal Glutathione redox states showed no significant differences between the cytosol and the peroxisome. This result was surprising since varieties were reported in literature before. Schwarzländer et al. (2015).